Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome

Background Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2). Methods We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Results Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. Conclusion As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype.


Background
Prader-Willi syndrome (PWS) is a complex neurodevelopmental disorder and a classic example for genomic imprinting in humans. The incidence is about 1 in 10-20,000, and the clinical manifestations include decreased fetal activity, neonatal hypotonia, neonatal feeding difficulties, hyperphagia with obesity, hypogonadism, short stature, small hands and feet, characteristic facial features, and mild to moderate mental retardation. Diagnostic criteria have been proposed [1] and revised recently [2].
About 70% of individuals clinically diagnosed with PWS have a ~4 Mb interstitial deletion at 15q11-13 of paternal origin, with clustered breakpoints (BP) at either of two proximal sites (BP1 or BP2) and one on the distal site (BP3) (Fig. 1a). The majority of the remainder have maternal uniparental disomy 15. In about 1 % of the cases, the disease is due to aberrant imprinting and gene silencing. Of these, 14% have small deletions in the imprinting center (IC) region of the paternal allele that abolish the expression of all imprinted paternally-expressed genes in cis. In the remainder no demonstrable DNA sequence changes have been observed [3][4][5][6][7]. In Angelman syndrome (AS, MIM#105830), which is usually caused by the same mechanisms affecting the maternal chromosome 15, mutations in the maternal copy of a single gene, UBE3A (MIM#601623), encoding a ubiquitin ligase, are detected in about 5% of cases, whereas in PWS, no disease-causing mutations in a single imprinted gene have yet been reported.
Multiple introns downstream of the SNURF-SNRPN coding region contain C/D box small nucleolar RNA (snoRNA) genes. There are two multi-copy snoRNA clusters (HBII-52 and PWCR1/HBII-85) [21,22], three single copy snoRNA genes (HBII-436, HBII-13, and HBII-437), and one snoRNA gene (HBII-438) present in two copies that are 240 kb apart [19]. Since the snoRNAs are derived from processed spliced-out introns, their expression is controlled by the SNRPN promoter and is highest in brain. The known function of other C/D box snoRNAs is to guide 2'-O -ribose methylation of ribosomal RNA or small nuclear RNA. This post-transcriptional modification is conserved throughout evolution and is thought to confer increased stability to the small RNA molecules [23]. The modification targets of the imprinted C/D box snoR-NAs in the PWS/AS region are still unknown.
Spontaneous chromosome translocations can be extremely valuable for assessing the contributions of individual loci to the phenotype of microdeletion syndromes. Five individuals with features of PWS have been reported who have balanced reciprocal translocations with breakpoints in the PWS/AS deletion region. All of them involve the SNRPN locus. The breakpoints are located in intron 2 (proximal, n = 2), disrupting the SNURF/SNRPN coding region, or in exon 20a/intron 20 (distal, n = 3) within the 3'-untranslated region of the long SNRPN transcript. One individual with a proximal and two of three patients with a distal breakpoint meet the diagnostic criteria for PWS (score of 8 or more points) [20,[24][25][26][27][28].
Here we report the clinical, cytogenetic and molecular characterization of a 22 year old male with features of PWS who has a different de novo balanced reciprocal translocation t(4;15)(q27;q11.2). We mapped the breakpoint to SNRPN intron 17 (position on chr 15: 22803227, UCSC Genome browser May 2004) and determined the expression of snoRNAs on both sides of the breakpoint in cultured fibroblasts and lymphoblasts.

Cytogenetic and FISH analysis
Metaphase spreads obtained from short-term blood lymphocyte cultures and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells (LCL) were processed for high-resolution GTG-banding by standard methods. For FISH studies, Bacterial Artificial Chromosomes (BACs) were sourced from the RPCI-11 library and selected using the UCSC Genome Browser, Assemblies: July 2003 and May 2004). Fluorescence labelling, hybridization procedures and imaging were performed as previously described [29].

DNA methylation study
Genomic DNA was purified by phenol-chloroform extraction from LCLs from the study subject, a normal control, and a PWS individual (Patient E in [6], Coriell Human Mutant Cell Repository # GM12134). To investigate methylation at exon 1 of SNRPN, 50 µg DNA were used for the bisulfite reaction and PCR with primers according to standard protocols [30,31]. PCR products were separated on a 3% agarose gel, stained with ethidium bromide, and visualized under UV illumination.

Expression studies by RT-PCR and quantitative RT-PCR
Total RNA was extracted from LCL and primary fibroblast cultures (FB) using RNA Stat 60. The RNA was treated with DNaseI (Roche) and RT-PCR was performed using Superscript II (Invitrogen). Primers were designed for exon-toexon amplification in an overlapping fashion -where possible -for SNRPN, MKRN3, MAGEL2, NDN, snoR-NAs, and two ESTs within Intron 20 of SNRPN (Table 1).
For a subset of exons in the SNPRN gene and the snoRNAs HBII-13, HBII-437, and PWCR1/HBII-85, quantitative Mapping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes Figure 1 Mapping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes . a . Schematic map of human chromosome region 15q11-q13. Black and gray circles represent imprinted genes, expressed from the paternal or maternal allele, respectively. White circles designate bi-allelically expressed genes. BP1, 2, and 3 indicate the locations of the deletion breakpoint hotspots [43]. b . FISH results placed BAC RP11-160D9 highlighted in green (nucleotide position 22577151-22735621) proximal to the translocation breakpoint and RP11-876N20 highlighted in blue (position 22857334-23036552) distal to the breakpoint. Intron 17, comprising nucleotides 22795282 to 22811656, thus is located ~ 63.4 kb downstream of RP11-160D9 and ~ 42 kb upstream of RP11-876N20. c . On representation of the SNRPN region (not drawn to scale) boxes represent exons and ESTs, lines represent snoRNA copies. Orange boxes and lines indicate exons, ESTs or snoR-NAs tested for expression either by RT-PCR or quantitative RT-PCR. Black flash indicates the breakpoint in intron 17 of the SNRPN locus.
RT-PCR assays were performed with SYBR Green I™ dye in an ABI 7700 cycler (Applied Biosystems) by using standard protocols [32,33]. Primers were designed to amplify products of 50 bp in length. GAPDH expression was used as a reference. Each sample was run at least in triplicate. The results were interpreted as described previously [28].
LCL RNA samples from a PWS individual with a microdeletion of the imprinting center (GM12134), a normal individual, an individual with an intrachromosomal triplication of the PWS region on the paternally-derived chromosome 15 (Patient 1 in [34], Coriell Human Mutant Cell Repository # GM12135), and fibroblast RNA from another t(4;15) PWS individual with the breakpoint in intron 2 of SNRPN [27,28] served as controls.

Southern blot analysis
Southern blot analysis was performed according to standard methods with ExpressHyb™ solution (BD Biosciences). Genomic DNA from a normal individual and the t(4;15) carrier was cleaved in a double digestion with restriction enzymes NheI and BsaWI to release a 6.4 kb fragment, and with NheI and ApaI to release a 10 kb fragment in the normal chromosome. The DNA probes were synthesized by PCR from genomic DNA and cloned into a pCRII T/A-vector (Invitrogen). The probes were designed to hybridize within intron 16 (SB-1) and upstream of the ApaI restriction site (SB-3) ( Table 1).

Breakpoint cloning with a PCR-based method
Genomic DNA from a normal individual and the t(4;15) carrier was cleaved in a double digestion with restriction enzymes EcoRV and ApaI, followed by adapter ligation according to the manufacturer's instructions (BD Genome Walker Universal Kit) [35]. A nested PCR reaction with adapter primers and sequence-specific primers was performed and the amplification products were cloned into the pC2.1 T/A-vector (Invitrogen) after gel purification. The clones were sequenced from both directions with universal primers from the vector (M13) and sequence specific primers.

Clinical case report
The patient (Fig. 2) was born at 41 weeks of gestation with a birth weight of 8 lb. Pregnancy was uneventful, but fetal movements were somewhat reduced. In the newborn, poor muscle tone, weak cry, excessive sleepiness, and undescended testes were observed. During infancy, he had poor suck and prolonged feeding times, but his weight gain was satisfactory and he did not require tube feeding.
He was suspected to have absence seizures of about 20 seconds duration, along with proneness to giggling, sometimes with eye-rolling. These episodes resolved by four years of age, and an EEG was normal.
He had a left esotropia that was surgically corrected. During childhood, sticky saliva, dry mouth, skin picking and a marked tolerance to pain were noted and have persisted.
Excessive daytime somnolence continued beyond infancy and treatment with amphetamine was started at 9 years of age. A sleep study, at 13

Cytogenetic analysis
High-resolution chromosome analysis showed an apparently balanced reciprocal translocation between the long arm of chromosome 4 and the proximal long arm of chromosome 15. The breakpoints were assigned to chromosome bands 4q27 and 15q11: 46, XY, t(4;15)(q27;q11) ( Fig. 3a). Parental chromosomes were normal, indicating that the patient's translocation was de novo.
a. High resolution G-banded ideograms and prometaphase chromosomes of the translocation derivatives and their normal homologs

DNA methylation analysis
To exclude alternative explanations for the phenotype, such as an imprinting defect, DNA methylation analysis was performed. Methylation-specific PCR of the SNURF-SNRPN exon 1 region revealed a normal bi-parental methylation pattern (Fig. 3b).

Mapping of the translocation breakpoint by FISH
We performed cytogenetic and molecular studies to characterize the breakpoint at 15q11 in detail. Preliminary FISH analysis showed that the breakpoint in 15q11 was located between D15S11 and GABRB3, which flank the SNRPN locus (data not shown). On this basis, a chromosome walking strategy was used across this region to narrow down the breakpoint region. We identified two BACs, RP11-160D9 (current position 22577151-22735621 on UCSC Genome Browser, May 2004 release) and RP11-876N20 (current position 22857334-23036552), that flanked the breakpoint and, thus, mapped it to a ~122 kb interval (Fig. 1b).

Fine mapping of the breakpoint by SNRPN expression and Southern blot analysis
To further refine the breakpoint, we carried out quantitative RT-PCR and RT-PCR experiments using RNA from an LCL and skin fibroblasts (FB) for expression of SNRPN transcripts. As shown in Figure 4 and Table 2, we found expression of SNPRN exons 2, 3, and 14 to 17, but no expression of exons 18 to 20, and concluded that the breakpoint falls within intron 17. For mapping intron 17, we designed a Southern blot using unique restriction sites. DNA cleaved with NheI and BsaWI showed a 6.4 kb band for the t(4;15) carrier and the normal control (Fig. 5b,  lanes 1 and 2), indicating that the breakpoint is located downstream of the BsaWI site. Samples doubly digested with NheI and ApaI (Fig. 5b, lanes 3-6), revealed additional bands for the translocation carrier. Besides the expected 10 kb band derived from the normal chromo-some 15, there was a ~11.5 kb band in lane 4, detected with the SB-1 probe, and a ~7 kb band in lane 6, detected with probe SB-3 (Fig. 5b). The novel ~11.5 kb band arose from the der(15) chromosome, with an NheI site on the chromosome 15 portion and an ApaI site on the chromosome 4 portion (Fig. 5c, upper panel). The novel band of 7 kb arose from the der(4) chromosome, with an ApaI site on the chromosome 15-part and an NheI site on the chromosome 4-part. Taken together, these results delimit the breakpoint region to ~3.6 kb between the BsaWI and ApaI sites (Fig. 5a).

Breakpoint mapping at the nucleotide level
By DNA sequencing, we mapped the breakpoint to SNRPN intron 17 (position chr 15: 22803227) and to chromosome 4 at position chr. 4:123965881 (UCSC Genome Browser, May 2004) (Fig. 6). On chromosome 4, a long terminal repeat (LTR) retrotransposon, LTR1B, spans the breakpoint. On chromosome 15, we found a short interspersed element (SINE), AluY, and a long interspersed element (LINE), L1M4, surrounding the breakpoint (Fig. 6a). Thirty-nine bp upstream of the breakpoint on chromosome 15 starts a common 26 bp core sequence of Alu elements (Alu-DEIN) in an inverted orientation. This sequence is known to be involved in gene rearrangements [36]. While the sequence across the breakpoint is contiguous on the der (15), an extra A is inserted on the der(4) chromosome (Fig. 6b). Furthermore, the breakpoint on chromosome 4 falls in a large intron between exons 10 and 11 of a spliced transcript (BC045668). By RT-PCR, we found that this transcript is expressed in fibroblasts, but not in LCLs (data not shown).

Expression of upstream genes MKRN3, MAGEL2, and NDN
Expression of the three imprinted genes MKRN3, MAGEL2, and NDN upstream of SNRPN was tested by RT- PCR in t(4;15) fibroblasts and found to be indistinguishable from expression in normal control fibroblasts (data not shown).

Expression of C/D box snoRNAs and intron-encoded ESTs
When testing for the intron-encoded C/D box snoRNAs, we were able to document expression of HBII-13 and HBII-437 and lack of expression for HBII-438A/B and HBII-85/PWCR1 (Fig. 7). By use of a more sensitive method, quantitative real-time RT-PCR, we obtained similar results for the SNRPN exons and snoRNAs tested ( Table 2). Two ESTs, AK094315 and AB061718 (= HBT8) located in the 30 kb SNRPN intron 20 were not expressed in the PWS control [6] and t(4;15) LCLs, but were expressed in the normal control LCL (Fig. 7).

Breakpoint mapping and mechanism of the translocation event
Dissecting the PWS deletion region and identifying individual genes as responsible for parts of the phenotype represent a challenge because all reported smaller deletions inactivate all imprinted genes on the paternally-derived chromosome 15. Rare reciprocal translocations, therefore, provide unique insights. We here report our studies of a 22 year old male with features of PWS who has a de novo SNRPN expression analysis by RT-PCR of RNA from LCLs balanced reciprocal translocation t(4;15)(q27;q11.2). This is the first such case where the translocation breakpoints have been identified at the DNA sequence level. The cytogenetic breakpoint designations in this individual are identical to those in another male PWS-like case with t(4;15)(q27;q11.2), previously reported by Kuslich and colleagues [27] and restudied by Gallagher and colleagues [28], which raised the intriguing possibility of a recurrent translocation that may be facilitated by genomic repeats or other distinct molecular features.
In the present case, however, we mapped the breakpoint to SNRPN intron 17 (position on chr. 15: 22803227, UCSC Genome Browser, May 2004) that differs from that in the previous case (SNRPN intron 2). Furthermore, the breakpoint in our case is novel as it does not fall into one Southern blot analysis identifies breakpoint in SNRPN intron 17 of the two previously described "breakpoint clusters" in intron 2 and exon 20a/intron 20 (Table 3 and Table 4). On chromosome 4 (chr. 4 123965881), the LTR retrotransposon LTR1B is spanning the breakpoint, and a short interspersed element (SINE), AluY, and a LINE element, L1M4, surround the breakpoint on chromosome 15 (Fig.  6). Interestingly, 39 bp upstream of the breakpoint on chromosome 15 starts a common 26-bp core sequence of Alu elements (Alu-DEIN) that has been shown to be involved in gene rearrangements and has homology with prokaryotic χ, an 8-bp sequence motif known to stimulate recBC mediated recombination in E. coli [37]. The core sequence is identical to sequences in the left arm of the consensus Alu element [38]. Sequence analyses of regions directly adjacent to translocation breakpoints has shown presence of the 26-bp Alu core sequence at or close (within 20-50 bp downstream or upstream) to the sites of recombination [36]. Therefore, this sequence might stimulate homologous and non-homologous recombination within the core or at nearby sites and could be the mechanism of recombination in the t(4;15) case reported here.
The translocation is de novo, as is true for all the previously described cases with translocation breakpoints involving the SNPRN gene. Given the PWS-like phenotype, the translocation was assumed to be of paternal origin. This Repeat sequences surrounding the breakpoint Figure 6 Repeat sequences surrounding the breakpoint . a . One hundred nucleotides on either side of the breakpoints on chromosome 4 and 15 contain repetitive sequences (grey lines). The Alu-DEIN sequence is located 13-39 bp upstream of the breakpoint on chromosome 15. b . Sequence across the breakpoint on the der(4) chromosome reveals an additional A inserted at the breakpoint. Arrows indicate the direction centromere to telomere.
assumption was confirmed by the expression studies. Paternal origin of the translocation was formally proven in 2 of the 5 previously reported cases [26,27].

Karyotype -phenotype correlations
Two individuals with SNRPN intron 2 breakpoints were described as having classical PWS, meeting the major clinical criteria by age 3.5 years and additional minor clinical criteria [25,27]. The individuals with a breakpoint in SNRPN Exon 20/Intron 20 were described as having a milder or atypical form of PWS (Table 3 and Table 4). The weight gain started later than in classical PWS, at 7 and 5 years, respectively, for the patients described by Schulze et al. 1996 andWirth et al. 2001, and at 8 years in our case. The characteristic facial features were absent in the case of Wirth et al. 2001, and also in the present case. But this is not a consistent feature in classical PWS, as in a retrospective evaluation of 90 molecularly-proven PWS cases, only 49% had the characteristic facial gestalt [39].
It is apparent from the review of the previously reported cases and the individual reported here (Table 3 and Table  4) that some of these translocation cases tend to have a milder, 'atypical' clinical picture, in comparison with classical PWS. There is not a complete absence of any of the major phenotypic features (neonatal hypotonia and feeding difficulty, hyperphagia from early childhood, obesity, cognitive compromise, hypogenitalism), but the degree of affection may be lower. None of the reported translocation cases had any additional features that might possibly be attributed to disruption of a gene on the reciprocal chromosome, and in no prior case had an attempt been made to identify a gene at this location.

Expression analysis in LCLs of snoRNAs and two ESTs in intron 20
Our sequence data mapped the breakpoint on chromosome 4 within intron 10 of a spliced polyadenylated transcript (BC045668). This unique cDNA clone represents a  unlikely that heterozygous disruption of this gene contributes to the phenotype in our patient.
Translocation has no effect on imprinting center methylation and upstream genes To assess whether the translocation event had affected the allele-specific methylation pattern at the imprinting center (IC), and/or to exclude a coincident imprinting defect, we carried out methylation studies of the SNRPN exon 1 region that revealed a normal bi-parental methylation pattern. Similar results were reported for each of the other five PWS individuals who had translocation breakpoints within the SNRPN gene. These results predict that expression of the genes located centromeric to the SNRPN exon 1/ IC region, NDN, MAGEL2 and MKRN3, should not be affected in these individuals. By studying t(4;15) fibroblasts by RT-PCR, we indeed found expression of all three genes. Previously, only MKRN3 was reported to be expressed in the three PWS translocation cases in which it was studied [20,25,27].
In t(4;15) lymphoblasts, the SNRPN transcript was detectable by RT-PCR and quantitative RT-PCR and found to extend all the way to exon 17. The major transcript that encodes the SNURF/SNRPN proteins terminates in exon 10 [20] and, therefore, should be unaffected by this t(4;15) translocation. With the caveat that studies on peripheral tissues, fibroblasts and lymphoblasts, may not accurately reflect gene expression in the brain, our results indicate that SNURF/SNRPN and the centromeric genes MKRN3, NDN and MAGEL2 are unlikely to play a prime role in the causation of PWS-associated features, although it remains an open question whether their loss or nonfunctioning might contribute to the more marked phenotypic expression that is seen in typical PWS.

Genes downstream of the breakpoint are not expressed
With respect to expression of downstream transcripts, the reported results on LCLs with breakpoints in exon 20/ intron 20 were consistent, whereas for the two patients with breakpoints in intron 2, the reported results were conflicting for expression of downstream transcripts IPW and PAR-1. In a re-evaluation of the t(4;15) case reported by Kuslich and colleagues [26], no expression of these transcripts and of the PWCR1/HBII-85 snoRNA cluster was detected by real-time quantitative RT-PCR [28].
Therefore, we focused our analysis on the snoRNAs and two ESTs in intron 20. As for the intron-encoded C/D box snoRNAs, HBII-13 and HBII-437 were expressed, and HBII-438A/B and HBII-85/PWCR1 were not. HBII-52 snoRNAs were not studied, as they are not expressed in the available tissues and have previously been excluded from contributing to the PWS phenotype [40,41]. The two ESTs in the large intron 20 that are highly expressed in brain tissues [42] were found to be expressed in a normal control LCL, but not in the t(4;15) LCL. This result suggests that these ESTs do not have their own promoter but are dependent on transcription from the SNRPN promoter that is located on the other translocation derivative in these cells. Therefore, these ESTs most likely represent stable derivatives of large alternatively spliced non-coding SNRPN transcripts.

Conclusion
(1) Expression of the ESTs and/or C/D box snoRNAs that are located downstream of the translocation breakpoint is not necessary for establishing and maintaining the paternal-specific pattern of gene expression pattern that is controlled by the imprinting center upstream of the translocation breakpoint.
(2) The C/D box snoRNAs HBII-438A and PWCR1/HBII-85 are the only stable transcripts in this region that are disrupted in this t(4;15) PWS individual. As PWCR1/HBII-85 sequences are highly conserved between human and mice, while no copy of HBII-438A has been found in mouse, we conclude that the basis of PWS pathogenesis resides, in whole or in part, in the absence of PWCR1/ HBII-85 snoRNA. SNURF/SNRPN and the centromeric genes MKRN3, NDN and MAGEL2 are unlikely to play a major role in the causation of PWS-associated features. While the function of known C/D box snoRNAs is to guide 2'-O -ribose methylation of mainly ribosomal RNA, these novel imprinted snoRNAs have no known target. They might be involved in a posttranscriptional regulation process of a gene or genes that -if non-functional -gives rise to the PWS phenotype.